Ebola virus is an emerging human pathogen that has been classified as a Category A priority virus. Ebola virus causes severe hemorrhagic fever and remains a threat as a potential agent of bioterrorism. Many of the molecular aspects of Ebola virus replication and pathogenesis remain ill defined, and there are currently no approved vaccines, nor antivirals to prevent or treat Ebola virus infections. Our laboratory was the first to demonstrate that the Ebola VP40 matrix protein can "bud" from mammalian cells in the absence of any other viral protein and in the form of a lipid containing, virus-like-particle (VLPs). Others and we have gone on to identify late budding domains (L-domains) within VP40 that facilitate VLP release by interacting with specific host proteins/machinery. While these Ebola VLP budding assays have been instrumental in enhancing our understanding of virus budding, the molecular mechanism and biological significance of L-domain function and host interactions during live Ebola virus infection remain to be elucidated. Our main hypothesis is that VP40 is the primary virus protein involved in budding, and that the function of VP40 is influenced and facilitated by in vivo interactions with both viral and cellular proteins. Therefore, a better understanding of VP40 function will provide insights into the molecular aspects of Ebola virus pathogenesis and may lead to the development of novel therapeutics to inhibit this late stage of Ebola virus replication. In this application, we will explore the use of inducible/stable cell lines, siRNA technology, and peptide transduction to ascertain the biological relevance of VP40 L-domains and host protein interactions in vivo. We will specifically: i) construct a VP40 inducible cell line to assess the contributions of additional viral (e.g. GP) and cellular (e.g. tsg101) proteins toward efficient VLP formation and release (Specific Aim 1). ii) establish the use of inducible siRNA cell lines to determine the significance of specific host proteins to Ebola virus budding. As proof-of-principle, we will construct an inducible cell line expressing a functional siRNA against tsg101 (Specific Aim 2). iii) establish the use of peptide transduction to introduce specific dominant-negative proteins into Ebola virus infected cells and mice to identify host proteins that are biologically relevant to the budding process of infectious Ebola virus. These studies will be fundamental for our understanding of Ebola virus budding and necessary for the successful development of future therapeutic approaches.